Macrophage migration inhibitory factor (MIF) and its homolog D-dopachrome tautomerase (D-DT) are significant promotors of UVB- but not chemically induced non-melanoma skin cancer

Non-melanoma skin cancer (NMSC) is the most common cancer in Caucasians worldwide. We investigated the pathophysiological role of MIF and its homolog D-DT in UVB- and chemically induced NMSC using Mif−/−, D-dt−/− and Mif−/−/D-dt−/− mice on a hairless SKH1 background. Knockout of both cytokines showed similar attenuating effects on inflammation after acute UVB irradiation and tumor formation during chronic UVB irradiation, without additive protective effects noted in double knockout mice, indicating that both cytokines activate a similar signaling threshold. In contrast, genetic deletion of Mif and D-dt had no major effects on chemically induced skin tumors. To get insight into the contributing mechanisms, we used an in vitro 3D skin model with incorporated macrophages. Application of recombinant MIF and D-DT led to an accumulation of macrophages within the epidermal part that could be reversed by selective inhibitors of MIF and D-DT pathways. In summary, our data indicate that MIF and D-DT contribute to the development and progression of UVB- but not chemically induced NMSC, a role at least partially accounted by effects of both cytokines on epidermal macrophage accumulation. These data highlight that MIF and D-DT are both potential therapeutic targets for the prevention of photocarcinogenesis but not chemical carcinogenesis.

www.nature.com/scientificreports/ fully understood, MIF has been shown to inhibit p53-dependent growth arrest and apoptosis to sustain activation responses 13,14 . MIF also has been described to be a pro-tumorigenic factor in various cancers that promotes angiogenesis, increases cell proliferation, and modulates tumor immunity [15][16][17] . The expression of MIF is increased in most solid and hematological malignancies and is often considered a negative prognostic indicator 8,18 . In this context, MIF may be a promising target for therapies 19,20 .
In the skin, high expression levels of MIF were found in the basal layer of the epidermis and cutaneous appendages 10,21 . Previous in vivo and in vitro studies revealed significant overexpression of MIF in cutaneous melanocytic tumors 19,22,23 . Using Mif −/− mice, a study by Martin et al. 13 indicated that MIF also plays an important role in the development and progression of UVB-induced NMSC. Our previous study revealed an enhanced expression of MIF in lesional skin of patients with actinic keratosis or cutaneous squamous cell carcinoma (SCC), together pointing towards an important role for MIF in the pathogenesis of NMSC 3 .
Various studies have revealed that D-dopachrome tautomerase (D-DT, also known as MIF-2) is a functional homolog of MIF, since both have a similar gene structure, 3D architecture, and enzyme activity 11,[24][25][26] . D-DT shares several biological activities with MIF and could therefore represent an endogenous amplifier of MIF action 21,27,28 ; however, divergent effects have also been reported 28,29 . Nevertheless, little is known about the biological function of D-DT and its role in photocarcinogenesis 30 . A previous study showed increased expression of D-DT in the epidermis of human skin after UVB exposure, similar to the expression of MIF, suggesting that D-DT may play a similar role as MIF in such inflammatory processes 31 . Recently, it was shown that chronic UVB exposure accelerates tumor development in D-dt-overexpressing transgenic mice, highlighting D-DT as a functionally important cytokine in photocarcinogenesis 32 .
The present study aimed to assess the pathophysiological function of MIF and its homolog D-DT in human cutaneous inflammatory reactions and the development and progression of UVB-and chemically induced NMSC. We determined the infiltration of Ly6G-positive neutrophils, as this is the hallmark of the acute inflammatory reaction (Fig. 1a). UVB-irradiated Mif −/− , D-dt −/− and Mif −/− /D-dt −/− mice exhibited significantly fewer infiltrating neutrophils than WT mice after 48 h (Fig. 1b). In addition, we measured the epidermal thickness of all mice and found that the epidermal thickness was significantly increased in UVB-irradiated Mif −/− and D-dt −/− mice compared to WT controls (Fig. 1c). Mif −/− /D-dt −/− mice also exhibited an increased epidermal thickness, but it was not significantly different when compared to the Mif −/− and D-dt −/− strains.

Figure 1.
Mifand D-dt deficient mice exhibit a significant decrease in inflammation after acute UVB exposure. All mice were dorsally irradiated with a single dose of UVB (2240 J/m 2 ) and dorsal skin was harvested 48 h later. (a) Representative Ly6G-stained sections of dorsal skin samples from UVB-irradiated mice (n = 6 mice per group). (b) Quantitative analysis of infiltrating neutrophils in UVB-exposed mice. Ly6G-expressing neutrophils were counted as cells per × 200 magnification, given as cells per high-power field (HPF) (c) Increase in epidermal thickness between UVB-irradiated mice versus non-irradiated mice. For each mouse epidermal thickness was measured at six different positions. Data represent mean ± SD. *p < 0.05; **p < 0.01. Scale bars = 200 µm. (d) qRT-PCR analysis of IL-6 expression in UVB-irradiated mice. (e) Increase in erythema index. Skin color for erythema assessment was measured before and 48 h after UVB irradiation. www.nature.com/scientificreports/ To further assess the severity of skin inflammation we measured erythema. Consistent with the lower neutrophil infiltration, all KO mice developed significantly less erythema compared to the WT controls (Fig. 1e).  (Fig. 2c). Consistently, all knockout groups displayed a significantly decreased tumor area per mouse compared to WT controls (Fig. 2d).

Delayed and suppressed tumorigenesis in
In addition, two randomly selected tumors from each mouse were assessed in blinded fashion by a dermatopathologist (Fig. 2e,f). While most tumors of WT mice were classified as SCC, those from each of the three KO mouse strains were predominantly papillomas.
Mif and D-dt deficiency does not influence tumor onset or the number of tumors, but leads to smaller tumors in a murine chemical skin carcinogenesis model. All mice displayed skin tumors after treatment with B(α)P twice weekly for 23 weeks (Fig. 3a). In contrast to our long-term UVB experiments, we did not observe any differences in the timing of tumor onset between each of the groups (Fig. 3b). First tumors of 1 mm 2 or larger appeared in each group at week 10 and all groups reached a tumor incidence of 100% at week 17. Consistent with these findings, the number of tumors induced by B(α)P was not statistically different between all groups in week 23 (  www.nature.com/scientificreports/ Two randomly selected tumors from each mouse were assesed in blinded fashion by a dermatohistopathologist ( Fig. 3e,f). The morphology of SCC in all groups appeared identical.
MIF and D-DT attract macrophages in the in vitro 3D skin model. As 3D skin models have proven to be a suitable tool to study the migration of macrophages through tissue 33 , we used such models and incorporated macrophages into the dermal equivalents to study the chemotactic effects of MIF and D-DT, respectively (Fig. 4a). Once the epidermal layer of the full-thickness skin models was stratified, recombinant human MIF, recombinant human D-DT, or both chemokines together were topically applied on the epidermal surface of the models. To further verify the specificity of the recruitment effects, additional models were treated topically with selective inhibitors of MIF (msR4M-L1) or D-DT (4-CPPC). 4-CPPC exhibits a 13-fold selectivity against D-DT over MIF 34,35 and msR4M-L1 has a fivefold higher affinity for MIF compared to D-DT 36 , and also is specific for MIF interactions with CXCR4 37 , a MIF recruitment receptor prominently expressed on macrophages.
Dermal skin from models was harvested 48 h after stimulation with the cytokines. In untreated control models the incorporated macrophages nearly disappeared (Fig. 4a). In contrast, stimulation with recombinant MIF, D-DT, and the combined treatment stopped the emigration of the macrophages from the models; macrophages even migrated in large numbers to the epidermal layers towards the stimulus of the topically applied cytokines (Fig. 4a upper row). These effects were reversed by additional treatment with the respective inhibitors, with only a small number of macrophages detectable in the dermis and almost no macrophages within the epidermal layers ( Fig. 4a lower row). The immunohistochemical observations were confirmed by quantitative measurements of CD68 + macrophages in the dermal and epidermal equivalents of the models (Fig. 4b,c).

Discussion
The most important environmental factor leading to skin cancer is UV radiation, which is estimated to be responsible for almost 90% of NMSCs 38,39 . UV exposure triggers acute inflammation in the skin by stimulating the production of proinflammatory cytokines, including MIF and its homolog D-DT 31,40,41 . The release of such cytokines (as well as neurotransmitters, endocrine factors, and neuropeptides) is a local effect after UVB exposure that can cause systemic effects leading to inflammatory diseases and malignancies 42,43 . www.nature.com/scientificreports/ Interesting but sometimes contradictory findings were made in previous studies on the roles of MIF and D-DT in skin tumors. Using Mif −/− BALB/c mice, Martin and colleagues 13 showed that MIF has tumor-promoting effects in chronic UVB-induced NMSC. Similar tumor-promoting characteristics were observed for D-DT when Yoshihisa et al. 32 showed that chronic UVB exposure accelerates tumor development in D-DT-overexpressing mice. Our group surprisingly classified MIF as a functional tumor suppressor in chemically-induced skin cancer models 10 . To our knowledge, we now present the first study to simultaneously investigate the role of MIF and D-DT in UVB-and chemically-induced NMSC by developing Mif −/− , D-dt −/− and Mif −/− /D-dt −/− mice on a hairless SKH1 background.
In our study, Mif −/− , D-dt −/− and Mif −/− /D-dt −/− mice exhibit a significantly reduced inflammatory response after acute UVB exposure as evidences by a reduction in neutrophil infiltration and erythema. These data confirm the previously reported inflammatory effects of MIF and D-DT during the acute response elicited in skin after UVB exposure 13,32 . Although a former study showed additive effects of MIF and D-DT on neutrophil recruitment to the lung 44 , we found that the deletion of only one of the two cytokines appears sufficient to reduce the inflammatory response in skin. In addition to tissue-specific reasons (lung versus skin), a mechanistic explanation for this observed discrepancy could be related to differences in cytokine administration/deletion. In the lung study, recombinant MIF or D-DT were locally and transiently administered, whereas in our current study, the global genetic deletion of Mif and D-dt may elicit secondary compensatory mechanisms.
IL-6 is a key player in inflammatory responses and it is associated with carcinogenesis 45 . Both MIF and D-DT have been shown to induce the expression of IL-6 46,47 . Consistently, the expression level of IL-6 mRNA was decreased in our KO mice after acute UVB exposure, reflecting the lower degree of inflammation in these mice. These observations may be related to the later onset and lower number of tumors in the KO mouse groups observed in our long-term UVB experiments.
Interestingly, in agreement with the data of Martin et al. 13 , we observed an increase of epidermal thickness in our KO mice after acute UVB irradiation. In contrast, Yoshihisa et al. 32 showed that D-DT overexpression in transgenic mice significantly increased epidermal thickness after acute UVB exposure. The authors further concluded that a D-DT-dependent increase in cell proliferation could be mediated via activation of the Akt signaling pathway 32   www.nature.com/scientificreports/ will be required to clarify the underlying mechanisms. However, it remains speculative that the increase in epidermal thickness plays a protective role and is a reason why the KO mice developed a lower tumor burden during long-term UVB exposure. There is broad consensus that MIF and D-DT often share similar roles to promote malignant transformation, tumor growth, and metastasis 30 . In our long-term UVB experiment, we found tumor-promoting effects for both cytokines that were consistent with former studies 13,32,48 . Only a small number of KO animals had developed an SCC compared to the WTcontrols. Putative additive effects of the DKO were not seen. A likely explanation is that MIF and D-DT are both involved in the same pathways. In fact, both cytokines can signal via CD74, the cognate MIF family receptor with key roles in tumorigenesis 49,50 , and recent preliminary evidence also hints at a role for CXCR4 as a shared receptor for MIF and D-DT 9,36 . Nevertheless, these results show that both cytokines are equally important in photocarcinogenesis and may be considered as potential therapeutic targets.
It is surprising that our previous study of chemically-induced skin carcinogenesis in mice of the 129Sv/ IMJ or C57Bl/6 backgrounds showed tumor-suppressive effects of Mif 10 . Using SKH1 mice in our present B(a) P-induced chemical carcinogenesis model, we found neither tumor-promoting nor tumor-suppressive effects of both cytokines, except that the KO animals showed a smaller tumor area. However, these results are at least consistent with the fact that neither cytokine has a tumor-promoting effect in non-UVB-induced skin cancer. Differences in immune cell recruitment between UVB-and chemically induced skin carcinogenesis could provide an explanation. UVB irradiation leads to multiple immunosuppressive effects that promote the formation of skin cancer 51 . In contrast, in chemical carcinogenesis, a significantly increased infiltration of leukocytes into the skin was observed 52 , which is likely to have an immunostimulatory effect. The fact that we did not observe tumor-suppressive effects of MIF in chemically induced NMSC could be explained by the choice of our mouse strain. It is likely that the SKH1 mouse strain may influence the outcome in our current chemically-induced skin cancer model, as we used only furry mice in our previous study. Although the mutation in the Hairless gene increases the susceptibility to UVB-induced tumorigenesis 53 , little is known about the use of SKH1 mice in chemically induced tumorigenesis models. Nevertheless, a few studies already proved that SKH1 mice are sensitive to chemically-induced skin cancer 54 . Thomas et al. 55 showed that SKH1 mice were highly prone to skin carcinogenesis with 7,12-dimethylbenz(a)anthracene (DMBA). The authors discussed that the lack of active hair follicles and the presence of abnormal hair follicles in these mice could indeed reduce the susceptibility to chemicals, but chemicals can still initiate carcinogenesis from the interfollicular epidermis or the rudimentary pilosebaceous appendages in these mice 55 . However, in addition to these anatomical features, SKH1 mice may also exhibit unknown genetic/molecular features that could explain our divergent results.
To get additional insight into the contributing mechanisms, we investigated the chemotactic effects of MIF and D-DT using an in vitro 3D skin model with incorporated macrophages. Topical application of MIF and D-DT to this model led to an accumulation of macrophages in the dermis and especially epidermis that could be reversed by selective MIF and D-DT inhibitors. The MIF inhibitor msR4M-L1 also bears specificity for the MIF/CXCR4 axis. Our findings are in line with studies showing that MIF and D-DT can induce the migration of monocytes and macrophages 9,56,57 . Although previous work has shown additive effects of MIF and D-DT on neutrophil recruitment 44 , our data do not support such additive chemotactic effects. This may be due to the notion that MIF-elicited migration of macrophages can also be mediated by CXCR2, whereas migratory responses induced by both MIF proteins involve interactions with CXCR4 9,28,36,37 , a receptor prominently expressed on both neutrophils and macrophages 58,59 . Nevertheless, our findings substantiate our previously postulated concept that MIF recruits cells of the innate immune system to the skin 10 . Our new data show this effect for the first time also for the MIF family member D-DT. Further investigations, especially on the topical application of MIF and D-DT inhibitors, also in combination with classic UV light protection preparations, would be useful for future in vitro and in vivo tests and perhaps also for later clinical use.
Taken together, we present the first study to investigate the role of MIF and its homolog D-DT in UVB-and chemically-induced NMSC using hairless Mif −/− , D-dt −/− and Mif −/− /D-dt −/− mice on a SKH1 background. Our data show that both cytokines have similar inflammatory effects after acute UVB exposure and tumor-promoting effects during chronic UVB irradiation. Observing no additive effects in DKO mice confirms that both cytokines activate a similar signaling threshold by the same receptor pathways. Interestingly, MIF and D-DT do not appear to have a major effect on chemically-induced skin tumors, which may be due to increased tumor immunity in chemical carcinogenesis. This indicates that both cytokines have only a limited tumor-promoting effect that can be neutralized by the immune system. Our data support the assumption that MIF and D-DT are both potential therapeutic targets for the prevention of photocarcinogenesis but not chemical carcinogenesis. www.nature.com/scientificreports/ mice (n = 5). Skin color for erythema assessment was measured with a Mexameter MX18 (Courage + Khazaka electronic GmbH, Cologne, Germany) immediately before UVB irradiation and after 48 h. The measurement is based on absorption/reflection. For the erythema measurement two specific wavelengths are used (green: 568 nm and red: 660 nm), corresponding to the spectral absorption peak of haemoglobin and to avoid other colour influences. The highly sensitive measurement gives values on a broad scale for erythema (0-999). All measurements were conducted in triplicates on the same skin area.

Animals. Ddt
Chronic UVB exposure. Each treatment group consisted of twelve mice (n = 12), while the corresponding non-irradiated control groups consisted of ten mice (n = 10 RNA isolation and quantitative real-time PCR analysis. RNA was isolated using the high-pure RNA isolation kit (Roche, Mannheim, Germany). RNA yield and purity were measured using a NanoDrop (Thermo, Erlangen, Germany). Purified RNA was reverse-transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Weiterstadt, Germany). TaqMan Gene Expression assays (Applied Biosystems) were used to study the quantitative expression of IL-6 (Hs00985641_m1) and Hprt (hypoxanthine phosphoribosyltransferase; Hs99999909_m1). Hprt was used as an internal reference to normalize the target transcript. All measurements were performed in triplicate. The qRT-PCR analyses were executed on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
Statistical analysis. Statistical analysis was performed using GraphPad PRISM version 7 (La Jolla, CA, USA). Values of *p < 0.05, **p < 0.01 and ***p < 0.001 were considered significant. Mann-Whitney U test was used to compare two groups. Percentage of tumor free mice was calculated and analyzed using the log-rank test. Comparison of tumor incidence was calaculated with the Χ 2 test.

Data availability
This paper does not include large-scale databases (next-generation sequencing or microarray). However, all data generated during and/ or analysed during these studies are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.